Impact of sex, MHC, and age of recipients on the therapeutic effect of transferred leukocytes from cancer-resistant SR/CR mice

To determine the impact of different recipient MHC and sex backgrounds on transferred anti-cancer activity, we performed a set of experiments where the mice were intentionally mismatched for strain and sex. For the sex-mismatch, SR/CR BALB/c male donor leukocytes were transferred into WT BALB/c female recipients, or SR/CR BALB/c female donor leukocytes were transferred into WT BALB/c males. For the MHC-mismatch, SR/CR BALB/c female donor leukocytes were transferred into WT C57BL/6 female recipients, or SR/CR BALB/c male donor leukocytes were transferred into WT C57BL/6 male recipients. For the sex- and MHC- double mismatch, SR/CR BALB/c male donor leukocytes were transferred into female WT C57BL/6 recipients, or SR/CR BALB/c female donor leukocytes were transferred into WT C57BL/6 males. All WT recipient mice were then challenged with 1 × 10⁶ S180 cells, 24 hours after the adoptive transfer, to evaluate the anticancer activity of the transferred leukocytes. The surviving mice were then challenged with 1 × 10⁶ S180 two more times with 5-6 week intervals between the injections. As expected, the sex- and MHC-matched controls showed 100% overall survival after all three S180 challenges (Figure 1). The sex-mismatch mice resulted in an overall survival of 83% (Figure 1) after three S180 challenges. The MHC-mismatch resulted in a reduction of overall survival (58%) which was maintained for each of the subsequent tumor challenges (Figure 1). When the recipients were mismatched for both MHC and sex, the overall survival dropped to approximately 42%, but remained unchanged after the first tumor challenge. Interestingly, when the recipients were evaluated by sex (Figure 2), the female recipients displayed a lower survival in all mismatched groups tested. When the transfers were mismatched for sex, 100% survival was observed in the male recipients with only 67% survival in the female recipients (Figure 2). When the recipients were MHC-mismatched, the male recipients had 83% survival while the females only had 33% survival (Figure 2). Finally, in the double-mismatch for sex and MHC, male recipients had a higher survival percentage (67%) when compared to the female recipients (17%) (Figure 2). All of the failed adoptive transfers were observed during the first challenge with S180 since the percent survival remained unchanged throughout the second and third challenges for all groups evaluated (Figure 1).

Out of the 36 mice that received mismatched adoptive transfers, 61% of the mice remained healthy with no signs of malignancy or GVHD development (Figure 3A, Column W). Following the first challenge with S180 cells, 31% of the recipients developed ascites (Figure 3A, Column X) which suggests that the donor leukocytes were rejected in the recipient. Approximately 5.5% of the mismatched mice died of unknown causes while approximately 2.5% of the mismatched mice (n = 1) displayed fur discoloration which is suggestive for GVHD (Figure 3A, Column Z, Figure 3B, C). This one mouse had a discoloration of fur (Figure 3B) that appeared approximately 14 days post-adoptive transfers. Upon histological evaluation, post inflammatory pigment incontinence was observed (Figure 3C). The black arrows point to areas with normal appearing pigmentation while the green arrows point to areas of pigment incontinence, in which the pigment was localized in macrophages at the periphery of the follicles. This observation suggests a prior inflammatory response that resolved prior to the end of the study. In the human setting, transfusion associated GVHD is a rare complication that is associated with fever, rash, abdominal pain, vomiting and diarrhea and is usually fatal [5, 6]. These symptoms were not found in the treated mice; however the fur discoloration is suggestive of a GVHD reaction.

Since most cancers occur in an aged population [10], we wanted to evaluate what impact this could have on adoptively transferred leukocytes. Leukocytes were transferred from young (4.75 ± 0.5 months) or old (22.88 ± 3.54 months) SR/CR C57BL/6 donors to young (3 months) or old (24 months) C57BL/6 WT mice, with sex and MHC are both matched. The recipient mice were then initially challenged with 1 × 10⁶ S180 24 hours after the adoptive transfer, followed by three subsequent S180 injections with 5-6 week intervals. As seen in the survival curve (Figure 4A), the control group (young donor leukocytes to young recipients) resulted in the highest overall survival percentage among the four groups. When an aged mouse was used as either the donor or the recipient, the survival was drastically reduced in all three groups, either immediately or over time following the adoptive transfer (Figure 4A). When the survival was evaluated after each S180 injection, three of the four groups (young donor to young recipient, old donor to old recipient, and old donor to young recipient) showed minimal changes after the initial S180 challenge (Figure 4B). In contrast to the other three groups, when young cells were transferred to an old environment, the recipients initially displayed a high percent survival, but then showed a continual decline after each subsequent challenge with S180 presumably due to the impact of the aged host environment on the donor leukocytes (Figure 4B).

The irradiation of cells can be used to help minimize the risk of GVHD in adoptive immunotherapy settings [12]. The irradiation irreversibly damages the DNA of the leukocytes, preventing their cell division without killing the cells. However, it is unclear if this process may have an immediate affect on the anticancer killing activity of the leukocytes. To evaluate this in our model, leukocytes of the spleen were harvested from C57BL/6 SR/CR mice, pooled, and then split into two groups. One group was irradiated (25Gy), while the other served as the nonirradiated control group. Each group was transferred to C57BL/6 WT mice and challenged 24 hours later with 1 × 10⁶S180. The nonirradiated controls resulted in 100% survival, while the irradiated group displayed a reduction to only 38% survival (Figure 5). Overall, irradiation negatively impacted, but did not abolish the cancer killing activity of the SR/CR leukocytes.

Cryopreservation is a common practice for storing cells [13, 14]. Most of the time the viability of stored cells is minimally affected by freezing and thawing, it is not clear whether a specific functionality, such as the anticancer activity of SR/CR donor leukocytes, can be affected by cryopreservation. Leukocytes were harvested from SR/CR donor mice, pooled and divided into two groups. One was given directly to WT mice as a non-frozen control, while the other cells were frozen for approximately 30 days. Then, these cells were thawed and given to another group of WT mice. There were 6 mice used for the post-cryopreservation due to the loss of a tube of cells during the retrieval process. By using 6 for the group, then the number of cells given per recipient was closer to the control recipients' numbers (within 2 × 10⁶ leukocytes). Both groups of WT mice were challenged with 1 × 10⁶ S180 cells 24 hours after the designated adoptive transfers. Even with the slightly reduced number of leukocytes delivered after cryopreservation, both groups showed identical survival (100%) after a challenge with S180 (Figure 5). Therefore cryopreservation appeared to have no negative impact on the cancer killing activity of the SR/CR leukocytes.

The S180 cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA). S180 cells were propagated in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in 5% carbon dioxide, or maintained by serial passage in wild-type (WT) C57BL/6 mice as ascites tumors. C57BL/6 (H-2b) and BALB/c (H-2d) mice were purchased from The Jackson Laboratory and Charles River, respectively. SR/CR mice in C57BL/6 (H-2b) and BALB/c (H-2d) backgrounds were bred at the animal resource program (ARP) facility of Wake Forest University School of Medicine. All mice were housed in plastic cages covered with individual air filter tops, containing corncob bedding, allowed free access to water and chow diet, and exposed to a 12-h fluorescent light/dark cycle. All procedures performed on the mice were in compliance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Wake Forest University and the National Institutes of Health Guide for the Care and Use of Animals.

For adoptive transfers, donor leukocytes were harvested from the spleens and bone marrow of sacrificed SR/CR mice, pooled in phosphate buffered saline and counted. The various experimental setups used one donor mouse per recipient with the exception of the mismatch experimental setup, which used one donor per recipient for the male donors and approximately 1.5 donors per recipient for the female donors due to differences in the spleen sizes. The number of leukocytes transferred to each donor was well above the minimum number of transferred leukocytes (5 × 10⁶) that are necessary to transfer the SR/CR phenotype (unpublished data). The number of transferred cells was matched within each experimental group based on a cell count or approximate spleen size and ranged from 51 × 10⁶ to 102 × 10⁶ or 1 to 1.5 spleens per recipient. The exception to this range and matching criteria were the cryopreservation adoptive transfer experimental group which had an approximately 2 × 10⁶ difference between the control and treated group (7-9 × 10⁶ for each recipient). The pooled donor leukocytes were then treated, if applicable, and injected intraperitoneally (IP) into recipient mice. One day after the leukocyte transfers, recipient mice were challenged with 1 × 10⁶ S180 to verify resistance. As previously shown [2], recipient mice survived the S180 challenges only if the transfers of the SR/CR leukocytes were successful.

When desired, leukocytes from donor mice were irradiated in flasks with a total accumulated dose of 25 Gray (Gy) with a cesium irradiator.

For cryopreserving, donor leukocytes were resuspended in a cryopreserving media consisting of 90% FBS +10% Dimethyl Sulfoxide (DMSO) and stored first in a -80°C freezer for approximately one day before being transferred to liquid nitrogen for approximately one month. Before transfers, the stored leukocytes were thawed in a 37°C water bath, washed two times with phosphate buffered saline to remove any remaining fetal bovine serum, and counted via trypan blue exclusion.

At desired time points of the studies, mice were sacrificed. The lungs, livers, intestines, kidneys, spleens and other tissues of potential interest were dissected and stored in 10% neutral buffered formalin. The collected tissues were embedded in paraffin. Sections of the tissues were stained with hematoxylin and eosin and examined for histological abnormalities.